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Jo, Areum, Sangwoo Ham, Gum Hwa Lee, Yun-Il Lee, SangSeong Kim, Yun-Song Lee, Joo-Ho Shin y Yunjong Lee. "Efficient Mitochondrial Genome Editing by CRISPR/Cas9". BioMed Research International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/305716.
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The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.
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Xie, Kabin, Bastian Minkenberg y Yinong Yang. "Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system". Proceedings of the National Academy of Sciences 112, n.º 11 (2 de marzo de 2015): 3570–75. http://dx.doi.org/10.1073/pnas.1420294112.
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The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.
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Mekler, Vladimir, Konstantin Kuznedelov y Konstantin Severinov. "Quantification of the affinities of CRISPR–Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences". Journal of Biological Chemistry 295, n.º 19 (1 de abril de 2020): 6509–17. http://dx.doi.org/10.1074/jbc.ra119.012239.
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The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.
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Bruegmann, Tobias, Khira Deecke y Matthias Fladung. "Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars". International Journal of Molecular Sciences 20, n.º 15 (24 de julio de 2019): 3623. http://dx.doi.org/10.3390/ijms20153623.
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CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.
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Wardhani, Bantari W. K., Meidi U. Puteri, Yukihide Watanabe, Melva Louisa, Rianto Setiabudy y Mitsuyasu Kato. "TMEPAI genome editing in triple negative breast cancer cells". Medical Journal of Indonesia 26, n.º 1 (16 de mayo de 2017): 14–8. http://dx.doi.org/10.13181/mji.v26i1.1871.
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Background: Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) is a powerful genome editing technique. It consists of RNA-guided DNA endonuclease Cas9 and single guide RNA (gRNA). By combining their expressions, high efficiency cleavage of the target gene can be achieved, leading to the formation of DNA double-strand break (DSB) at the genomic locus of interest which will be repaired via NHEJ (non-homologous end joining) or HDR (homology-directed repair) and mediate DNA alteration. We aimed to apply the CRISPR/Cas9 technique to knock-out the transmembrane prostate androgen-induced protein (TMEPAI) gene in the triple negative breast cancer cell line.Methods: Designed gRNA which targets the TMEPAI gene was synthesized, annealed, and cloned into gRNA expression vector. It was co-transfected into the TNBC cell line using polyethylenimine (PEI) together with Cas9-GFP and puromycin resistant gene vector. At 24-hours post-transfection, cells were selected by puromycin for 3 days before they were cloned. Selected knock-out clones were subsequently checked on their protein levels by western blotting.Results: CRISPR/Cas9, a genome engineering technique successfully knocked-out TMEPAI in the Hs578T TNBC cell line. Sequencing shows a frameshift mutation in TMEPAI. Western blot shows the absence of TMEPAI band on Hs578T KO cells.Conclusion: TMEPAI gene was deleted in the TNBC cell line using the genomic editing technique CRISPR/Cas9. The deletion was confirmed by genome and protein analysis.
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Kong, Qihui, Jie Li, Shoudong Wang, Xianzhong Feng y Huixia Shou. "Combination of Hairy Root and Whole-Plant Transformation Protocols to Achieve Efficient CRISPR/Cas9 Genome Editing in Soybean". Plants 12, n.º 5 (23 de febrero de 2023): 1017. http://dx.doi.org/10.3390/plants12051017.
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The new gene-editing technology CRISPR/Cas system has been widely used for genome engineering in various organisms. Since the CRISPR/Cas gene-editing system has a certain possibility of low efficiency and the whole plant transformation of soybean is time-consuming and laborious, it is important to evaluate the editing efficiency of designed CRISPR constructs before the stable whole plant transformation process starts. Here, we provide a modified protocol for generating transgenic hairy soybean roots to assess the efficiency of guide RNA (gRNA) sequences of the CRISPR/Cas constructs within 14 days. The cost- and space-effective protocol was first tested in transgenic soybean harboring the GUS reporter gene for the efficiency of different gRNA sequences. Targeted DNA mutations were detected in 71.43–97.62% of the transgenic hairy roots analyzed as evident by GUS staining and DNA sequencing of the target region. Among the four designed gene-editing sites, the highest editing efficiency occurred at the 3′ terminal of the GUS gene. In addition to the reporter gene, the protocol was tested for the gene-editing of 26 soybean genes. Among the gRNAs selected for stable transformation, the editing efficiency of hairy root transformation and stable transformation ranged from 5% to 88.8% and 2.7% to 80%, respectively. The editing efficiencies of stable transformation were positively correlated with those of hairy root transformation with a Pearson correlation coefficient (r) of 0.83. Our results demonstrated that soybean hairy root transformation could rapidly assess the efficiency of designed gRNA sequences on genome editing. This method can not only be directly applied to the functional study of root-specific genes, but more importantly, it can be applied to the pre-screening of gRNA in CRISPR/Cas gene editing.
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Jameel, Mohd Rizwan. "From design to validation of CRISPR/gRNA primers towards genome editing". Bioinformation 18, n.º 5 (31 de mayo de 2022): 471–77. http://dx.doi.org/10.6026/97320630018471.
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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated system) is used to edit specific genomic sequences with precision and efficacy. There are many online platforms/software for the design of gRNAs and related primers. However, there are concerns in design regarding off-site deletions besides knocking out sequences in the target genes. Nonetheless, a well known robust platform for CRISPR/gRNA primers design is CRISPRdirect. We demonstrate the use of this tool in the design of CRISPR/gRNA primers for soluble starch synthases (SSS) II-1, 2, and 3 genes in the Oryza sativa genome followed by the PCR-mediated amplification of SSS genes with corresponding confirmation towards genome editing having improved phenotype features.
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Jung, Soo Bin, Chae young Lee, Kwang-Ho Lee, Kyu Heo y Si Ho Choi. "A cleavage-based surrogate reporter for the evaluation of CRISPR–Cas9 cleavage efficiency". Nucleic Acids Research 49, n.º 15 (4 de junio de 2021): e85-e85. http://dx.doi.org/10.1093/nar/gkab467.
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Abstract CRISPR–Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1α promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR–Cas9 cleavage at a target site located between the EF-1α promoter and the lacI gene, repressor expression is disrupted, thereby triggering luciferase or EGFP expression. Using this system, we can quantitate gRNA cleavage efficiency by assessing luciferase activity or EGFP expression. We found a strong positive correlation between the cleavage efficiency of gRNAs measured using this reporter and mutation frequency, measured using surveyor and deep sequencing. The genome-editing efficiency of gRNAs was validated in human liver organoids. Our LacI-reporter system provides a useful tool to select efficient gRNAs for genome editing.
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Foreman, Hui-Chen Chang, Varvara Kirillov, Gabrielle Paniccia, Demetra Catalano, Trevor Andrunik, Swati Gupta, Laurie T. Krug y Yue Zhang. "RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production". PLOS ONE 16, n.º 6 (4 de junio de 2021): e0252313. http://dx.doi.org/10.1371/journal.pone.0252313.
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Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.
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Yoo, Byung-Chun, Narendra S. Yadav, Emil M. Orozco y Hajime Sakai. "Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts". PeerJ 8 (6 de enero de 2020): e8362. http://dx.doi.org/10.7717/peerj.8362.
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We present a new approach to edit both mitochondrial and chloroplast genomes. Organelles have been considered off-limits to CRISPR due to their impermeability to most RNA and DNA. This has prevented applications of Cas9/gRNA-mediated genome editing in organelles while the tool has been widely used for engineering of nuclear DNA in a number of organisms in the last several years. To overcome the hurdle, we designed a new approach to enable organelle genome editing. The plasmids, designated “Edit Plasmids,” were constructed with two expression cassettes, one for the expression of Cas9, codon-optimized for each organelle, under promoters specific to each organelle, and the other cassette for the expression of guide RNAs under another set of promoters specific to each organelle. In addition, Edit Plasmids were designed to carry the donor DNA for integration between two double-strand break sites induced by Cas9/gRNAs. Each donor DNA was flanked by the regions homologous to both ends of the integration site that were short enough to minimize spontaneous recombination events. Furthermore, the donor DNA was so modified that it did not carry functional gRNA target sites, allowing the stability of the integrated DNA without being excised by further Cas9/gRNAs activity. Edit Plasmids were introduced into organelles through microprojectile transformation. We confirmed donor DNA insertion at the target sites facilitated by homologous recombination only in the presence of Cas9/gRNA activity in yeast mitochondria and Chlamydomonas chloroplasts. We also showed that Edit Plasmids persist and replicate in mitochondria autonomously for several dozens of generations in the presence of the wild-type genomes. Finally, we did not find insertions and/or deletions at one of the Cas9 cleavage sites in Chloroplasts, which are otherwise hallmarks of Cas9/gRNA-mediated non-homologous end joining (NHEJ) repair events in nuclear DNA. This is consistent with previous reports of the lack of NHEJ repair system in most bacteria, which are believed to be ancestors of organelles. This is the first demonstration of CRISPR-mediated genome editing in both mitochondria and chloroplasts in two distantly related organisms. The Edit Plasmid approach is expected to open the door to engineer organelle genomes of a wide range of organisms in a precise fashion.
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Cheng, Hao, Feng Zhang y Yang Ding. "CRISPR/Cas9 Delivery System Engineering for Genome Editing in Therapeutic Applications". Pharmaceutics 13, n.º 10 (9 de octubre de 2021): 1649. http://dx.doi.org/10.3390/pharmaceutics13101649.
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The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) systems have emerged as a robust and versatile genome editing platform for gene correction, transcriptional regulation, disease modeling, and nucleic acids imaging. However, the insufficient transfection and off-target risks have seriously hampered the potential biomedical applications of CRISPR/Cas9 technology. Herein, we review the recent progress towards CRISPR/Cas9 system delivery based on viral and non-viral vectors. We summarize the CRISPR/Cas9-inspired clinical trials and analyze the CRISPR/Cas9 delivery technology applied in the trials. The rational-designed non-viral vectors for delivering three typical forms of CRISPR/Cas9 system, including plasmid DNA (pDNA), mRNA, and ribonucleoprotein (RNP, Cas9 protein complexed with gRNA) were highlighted in this review. The vector-derived strategies to tackle the off-target concerns were further discussed. Moreover, we consider the challenges and prospects to realize the clinical potential of CRISPR/Cas9-based genome editing.
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Sant’Ana, Rodrigo Ribeiro Arnt, Clarissa Alves Caprestano, Rubens Onofre Nodari y Sarah Zanon Agapito-Tenfen. "PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts". Genes 11, n.º 9 (2 de septiembre de 2020): 1029. http://dx.doi.org/10.3390/genes11091029.
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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated ones, especially plant cells, is still challenging. In a protoplast system, previous studies claimed that such pitfalls would be averted by delivering pre-assembled ribonucleoprotein complexes (RNPs) composed of purified recombinant Cas9 enzyme and in vitro transcribed guide RNA (gRNA) molecules. We, therefore, aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with polyethylene glycol (PEG) 4000. We performed an effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene, and by applying two different exposure times to RNPs. Using a low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between the exposure time and mutation frequency. The mutation frequency was gRNA sequence- and exposure time-dependent. In the present study, we demonstrated that the suitability of RNP transfection was proven as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for the selection of gRNA suitable for the editing of the gene of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively. Nevertheless, the large amplitude of mutations at the target site require scrutiny when checking mutations at off-target sites and potential safety concerns.
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Bhagwat, Aditya M., Johannes Graumann, Rene Wiegandt, Mette Bentsen, Jordan Welker, Carsten Kuenne, Jens Preussner, Thomas Braun y Mario Looso. "multicrispr: gRNA design for prime editing and parallel targeting of thousands of targets". Life Science Alliance 3, n.º 11 (9 de septiembre de 2020): e202000757. http://dx.doi.org/10.26508/lsa.202000757.
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Targeting the coding genome to introduce nucleotide deletions/insertions via the CRISPR/Cas9 technology has become a standard procedure. It has quickly spawned a multitude of methods such as prime editing, APEX proximity labeling, or homology directed repair, for which supporting bioinformatics tools are, however, lagging behind. New CRISPR/Cas9 applications often require specific gRNA design functionality, and a generic tool is critically missing. Here, we introduce multicrispr, an R/bioconductor tool, intended to design individual gRNAs and complex gRNA libraries. The package is easy to use; detects, scores, and filters gRNAs on both efficiency and specificity; visualizes and aggregates results per target or CRISPR/Cas9 sequence; and finally returns both genomic ranges and sequences of gRNAs. To be generic, multicrispr defines and implements a genomic arithmetic framework as a basis for facile adaptation to techniques recently introduced such as prime editing or yet to arise. Its performance and design concepts such as target set–specific filtering render multicrispr a tool of choice when dealing with screening-like approaches.
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Ji, Jie, Chunyang Zhang, Zhongfeng Sun, Longlong Wang, Deqiang Duanmu y Qiuling Fan. "Genome Editing in Cowpea Vigna unguiculata Using CRISPR-Cas9". International Journal of Molecular Sciences 20, n.º 10 (19 de mayo de 2019): 2471. http://dx.doi.org/10.3390/ijms20102471.
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Cowpea (Vigna unguiculata) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in Vigna unguiculata is dramatically hampered by the lack of mutant resources and efficient tools for gene inactivation in vivo. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We applied the CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the representative symbiotic nitrogen fixation (SNF) gene in Vigna unguiculata. Our customized guide RNAs (gRNAs) targeting symbiosis receptor-like kinase (SYMRK) achieved ~67% mutagenic efficiency in hairy-root-transformed plants, and nodule formation was completely blocked in the mutants with both alleles disrupted. Various types of mutations were observed near the PAM region of the respective gRNA. These results demonstrate the applicability of the CRISPR/Cas9 system in Vigna unguiculata, and therefore should significantly stimulate functional genomics analyses of many important agronomical traits in this unique crop legume.
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Easmin, Farhana, Naim Hassan, Yu Sasano, Keisuke Ekino, Hisataka Taguchi y Satoshi Harashima. "gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing". Journal of Bioscience and Bioengineering 128, n.º 3 (septiembre de 2019): 373–78. http://dx.doi.org/10.1016/j.jbiosc.2019.02.009.
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Alok, Anshu, Hanny Chauhan, Santosh Kumar Upadhyay, Ashutosh Pandey, Jitendra Kumar y Kashmir Singh. "Compendium of Plant-Specific CRISPR Vectors and Their Technical Advantages". Life 11, n.º 10 (28 de septiembre de 2021): 1021. http://dx.doi.org/10.3390/life11101021.
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CRISPR/Cas mediated genome editing is a revolutionary approach for manipulating the plant genome. However, the success of this technology is highly dependent on selection of a specific vector and the other components. A plant-specific CRISPR/Cas vector usually consists of a Cas gene, target-specific gRNA, leader sequence, selectable marker gene, precise promoters, and other accessories. It has always been challenging to select the specific vector for each study due to a lack of comprehensive information on CRISPR vectors in one place. Herein, we have discussed every technical aspect of various important elements that will be highly useful in vector selection and efficient editing of the desired plant genome. Various factors such as the promoter regulating the expression of Cas and gRNA, gRNA size, Cas variants, multicistronic gRNA, and vector backbone, etc. influence transformation and editing frequency. For example, the use of polycistronic tRNA-gRNA, and Csy4-gRNA has been documented to enhance the editing efficiency. Similarly, the selection of an efficient selectable marker is also a very important factor. Information on the availability of numerous variants of Cas endonucleases, such as Cas9, Cas12a, Cas12b, Casɸ, and CasMINI, etc., with diverse recognition specificities further broadens the scope of editing. The development of chimeric proteins such as Cas fused to cytosine or adenosine deaminase domain and modified reverse transcriptase using protein engineering enabled base and prime editing, respectively. In addition, the newly discovered Casɸ and CasMINI would increase the scope of genetic engineering in plants by being smaller Cas variants. All advancements would contribute to the development of various tools required for gene editing, targeted gene insertion, transcriptional activation/suppression, multiplexing, prime editing, base editing, and gene tagging. This review will serve as an encyclopedia for plant-specific CRISPR vectors and will be useful for researchers.
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Gasanov, Eugene V., Justyna Jędrychowska, Michal Pastor, Malgorzata Wiweger, Axel Methner y Vladimir P. Korzh. "An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique". Molecular Biology Reports 48, n.º 2 (22 de enero de 2021): 1951–57. http://dx.doi.org/10.1007/s11033-020-06125-8.
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AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.
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Yamamoto, Akihiro, Takashi Ishida, Mika Yoshimura, Yuri Kimura y Shinichiro Sawa. "Developing Heritable Mutations in Arabidopsis thaliana Using a Modified CRISPR/Cas9 Toolkit Comprising PAM-Altered Cas9 Variants and gRNAs". Plant and Cell Physiology 60, n.º 10 (14 de junio de 2019): 2255–62. http://dx.doi.org/10.1093/pcp/pcz118.
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Abstract Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), comprising an RNA-guided DNA endonuclease and a programmable guide RNA (gRNA), is currently recognized to be a powerful genome-editing tool and is widely used in biological science. Despite the usefulness of the system, a protospacer-adjacent motif (PAM) immediately downstream of the target sequence needs to be taken into account in the design of the gRNA, a requirement which limits the flexibility of the CRISPR-based genome-editing system. To overcome this limitation, a Cas9 isolated from Streptococcus pyogenes, namely SpCas9, engineered to develop several variants of Cas9 nuclease, has been generated. SpCas9 recognizes the NGG sequence as the PAM, whereas its variants are capable of interacting with different PAMs. Despite the potential advantage of the Cas9 variants, their functionalities have not previously been tested in the widely used model plant, Arabidopsis thaliana. Here, we developed a plant-specific vector series harboring SpCas9-VQR (NGAN or NGNG) or SpCas9-EQR (NGAG) and evaluated their functionalities. These modified Cas9 nucleases efficiently introduced mutations into the CLV3 and AS1 target genes using gRNAs that were compatible with atypical PAMs. Furthermore, the generated mutations were passed on to their offspring. This study illustrated the usefulness of the SpCas9 variants because the ability to generate heritable mutations will be of great benefit in molecular genetic analyses. A greater number of potential SpCas9-variant-recognition sites in these genes are predicted, compared with those of conventional SpCas9. These results demonstrated the usefulness of the SpCas9 variants for genome editing in the field of plant science research.
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Lessard, Samuel, Laurent Francioli, Jessica Alfoldi, Jean-Claude Tardif, Patrick T. Ellinor, Daniel G. MacArthur, Guillaume Lettre, Stuart H. Orkin y Matthew C. Canver. "Human genetic variation alters CRISPR-Cas9 on- and off-targeting specificity at therapeutically implicated loci". Proceedings of the National Academy of Sciences 114, n.º 52 (11 de diciembre de 2017): E11257—E11266. http://dx.doi.org/10.1073/pnas.1714640114.
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The CRISPR-Cas9 nuclease system holds enormous potential for therapeutic genome editing of a wide spectrum of diseases. Large efforts have been made to further understanding of on- and off-target activity to assist the design of CRISPR-based therapies with optimized efficacy and safety. However, current efforts have largely focused on the reference genome or the genome of cell lines to evaluate guide RNA (gRNA) efficiency, safety, and toxicity. Here, we examine the effect of human genetic variation on both on- and off-target specificity. Specifically, we utilize 7,444 whole-genome sequences to examine the effect of variants on the targeting specificity of ∼3,000 gRNAs across 30 therapeutically implicated loci. We demonstrate that human genetic variation can alter the off-target landscape genome-wide including creating and destroying protospacer adjacent motifs (PAMs). Furthermore, single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) can result in altered on-target sites and novel potent off-target sites, which can predispose patients to treatment failure and adverse effects, respectively; however, these events are rare. Taken together, these data highlight the importance of considering individual genomes for therapeutic genome-editing applications for the design and evaluation of CRISPR-based therapies to minimize risk of treatment failure and/or adverse outcomes.
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Westbrook, Adam W., Murray Moo-Young y C. Perry Chou. "Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis". Applied and Environmental Microbiology 82, n.º 16 (3 de junio de 2016): 4876–95. http://dx.doi.org/10.1128/aem.01159-16.
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ABSTRACTThe establishment of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system for strain construction inBacillus subtilisis essential for its progression toward industrial utility. Here we outline the development of a CRISPR-Cas9 tool kit for comprehensive genetic engineering inB. subtilis. In addition to site-specific mutation and gene insertion, our approach enables continuous genome editing and multiplexing and is extended to CRISPR interference (CRISPRi) for transcriptional modulation. Our tool kit employs chromosomal expression of Cas9 and chromosomal transcription of guide RNAs (gRNAs) using a gRNA transcription cassette and counterselectable gRNA delivery vectors. Our design obviates the need for multicopy plasmids, which can be unstable and impede cell viability. Efficiencies of up to 100% and 85% were obtained for single and double gene mutations, respectively. Also, a 2.9-kb hyaluronic acid (HA) biosynthetic operon was chromosomally inserted with an efficiency of 69%. Furthermore, repression of a heterologous reporter gene was achieved, demonstrating the versatility of the tool kit. The performance of our tool kit is comparable with those of systems developed forEscherichia coliandSaccharomyces cerevisiae, which rely on replicating vectors to implement CRISPR-Cas9 machinery.IMPORTANCEIn this paper, as the first approach, we report implementation of the CRISPR-Cas9 system inBacillus subtilis, which is recognized as a valuable host system for biomanufacturing. The study enables comprehensive engineering ofB. subtilisstrains with virtually any desired genotypes/phenotypes and biochemical properties for extensive industrial application.
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Ghoshal, Basudev, Brandon Vong, Colette L. Picard, Suhua Feng, Janet M. Tam y Steven E. Jacobsen. "A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation in Arabidopsis thaliana". PLOS Genetics 16, n.º 12 (14 de diciembre de 2020): e1008983. http://dx.doi.org/10.1371/journal.pgen.1008983.
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Plant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation of the FLOWERING WAGENINGEN gene in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.
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Aiba, Wataru, Takamitsu Amai, Mitsuyoshi Ueda y Kouichi Kuroda. "Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases". Biomolecules 12, n.º 11 (3 de noviembre de 2022): 1621. http://dx.doi.org/10.3390/biom12111621.
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In precise genome editing, site-specific DNA double-strand breaks (DSBs) induced by the CRISPR/Cas9 system are repaired via homology-directed repair (HDR) using exogenous donor DNA templates. However, the low efficiency of HDR-mediated genome editing is a barrier to widespread use. In this study, we created a donor DNA/guide RNA (gRNA) hybrid duplex (DGybrid) that was composed of sequence-extended gRNA and single-stranded oligodeoxynucleotide (ssODN) combined with complementary bases without chemical modifications to increase the concentration of donor DNA at the cleavage site. The efficiency of genome editing using DGybrid was evaluated in Saccharomyces cerevisiae. The results show a 1.8-fold (from 35% to 62%) improvement in HDR-mediated editing efficiency compared to genome editing in which gRNA and donor DNA were introduced separately. In addition, analysis of the nucleic acid introduction efficiency using flow cytometry indicated that both RNA and ssODNs are efficiently incorporated into cells together by using the DNA/RNA hybrid. Our technique would be preferred as a universal and concise tool for improving the efficiency of HDR-mediated genome editing.
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Traxler, Elizabeth, Yu Yao, Chunliang Li, Jeremy Grevet, Peng Huang, Shaela Wright, Gerd A. Blobel y Mitchell J. Weiss. "Genome Editing Recreates Hereditary Persistence of Fetal Hemoglobin in Primary Human Erythroblasts". Blood 126, n.º 23 (3 de diciembre de 2015): 640. http://dx.doi.org/10.1182/blood.v126.23.640.640.
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Abstract Manipulating the developmental switch from γ- to β-globin expression that occurs after birth has been intensively investigated as therapeutic strategy for sickle cell anemia and β-thalassemia. Rare individuals with a benign condition termed hereditary persistence of fetal hemoglobin (HPFH) exhibit an attenuated or absent γ-to-β switch, resulting in high levels of fetal hemoglobin (α2γ2) in all red blood cells (RBCs) throughout life. Moreover, individuals with HPFH and homozygosity for sickle cell disease (SCD) mutations exhibit few or no clinical manifestations of the latter. We used genome editing to induce a naturally occurring 13-nucleotide (-102 to -114) deletional HPFH mutation in the γ-globin (HBG1) gene promoter. Heterozygosity for this mutation is associated with HbF levels > 30% in adults. We used the clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system to create small deletions around -102 to -114 in the γ-globin genes in peripheral blood CD34+ cells from healthy donors. We delivered guide RNA (gRNA) and Cas9 using lentiviruses, sorted transduced hematopoietic progenitors by FACS, and cultured them using a 3-phase erythroid differentiation protocol. Real time PCR showed that γ-globin mRNA increased more than 10-fold in Cas9/gRNA transduced cells compared to controls. HbF flow cytometry and high-performance liquid chromatography (HPLC) demonstrated that induced γ-globin chains were effectively incorporated into hemoglobin tetramers. HPLC revealed 1-3% HbF in negative controls and an increase to 15% in cells transduced with gRNA and Cas9. Expression of erythroid differentiation markers CD235 and CD71 were unaffected, suggesting that the γ-globin increase is not due to impaired erythroid maturation. Next generation sequencing demonstrated that a single gRNA created one predominant mutation that co-segregated with high HbF expression and represented over 50% of the sequencing coverage. Interestingly, this mutation is identical to the 13-nucleotide HPFH deletion. We also tested the gRNA mutation efficiency after transient expression of gRNA and Cas9 in human CD34+ cells by electroporation followed by analysis of single burst-forming unit-erythroid (BFU-E) colonies formed in methylcellulose. Genomic DNA analysis revealed that one gRNA targeted 50% of HBG1 alleles, and cells that received two overlapping gRNAs demonstrated 80% mutation frequency. Real-time PCR of mRNA from edited BFU-Es showed that mutations stimulated γ-globin mRNA expression to 19-55% total globin synthesis, whereas control colonies contained 1-5% γ-globin. Together, our data demonstrate that the CRISPR-Cas9 system can generate precisely the -102 to -114 HPFH mutation at high efficiency in primary human progenitor cells and thereby induce the expression of HbF to potentially therapeutic levels. This work provides proof of concept for targeted genome editing for γ-globin activation as a therapy for patients with β hemoglobinopathies. Disclosures Weiss: Biogen: Research Funding; GlaxoSmithKline: Consultancy; Rubius: Membership on an entity's Board of Directors or advisory committees.
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Gao, Zongliang, Minghui Fan, Atze T. Das, Elena Herrera-Carrillo y Ben Berkhout. "Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA". Nucleic Acids Research 48, n.º 10 (13 de abril de 2020): 5527–39. http://dx.doi.org/10.1093/nar/gkaa226.
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Abstract The CRISPR-Cas9 system has been used for genome editing of various organisms. We reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections with a single guide RNA (gRNA) and subsequent viral escape, but complete inactivation of infectious HIV with certain combinations of two gRNAs. The new RNA-guided endonuclease system CRISPR-Cas12a (formerly Cpf1) may provide a more promising tool for genome engineering with increased activity and specificity. We compared Cas12a to the original Cas9 system for inactivation of the integrated HIV DNA genome. Superior antiviral activity is reported for Cas12a, which can achieve full HIV inactivation with only a single gRNA (called crRNA). We propose that the different architecture of Cas9 versus Cas12a endonuclease explains this effect. We also disclose that DNA cleavage by the Cas12a endonuclease and subsequent DNA repair causes mutations with a sequence profile that is distinct from that of Cas9. Both CRISPR systems can induce the typical small deletions around the site of DNA cleavage and subsequent repair, but Cas12a does not induce the pure DNA insertions that are routinely observed for Cas9. Although these typical signatures are apparent in many literature studies, this is the first report that documents these striking differences.
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Binyameen, Barkha, Zulqurnain Khan, Sultan Habibullah Khan, Aftab Ahmad, Nayla Munawar, Muhammad Salman Mubarik, Hasan Riaz et al. "Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus". International Journal of Molecular Sciences 22, n.º 22 (21 de noviembre de 2021): 12543. http://dx.doi.org/10.3390/ijms222212543.
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In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3–5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20–40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60−70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production.
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Roberson, Elisha D. "Identification of high-efficiency 3′GG gRNA motifs in indexed FASTA files with ngg2". PeerJ Computer Science 1 (18 de noviembre de 2015): e33. http://dx.doi.org/10.7717/peerj-cs.33.
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CRISPR/Cas9 is emerging as one of the most-used methods of genome modification in organisms ranging from bacteria to human cells. However, the efficiency of editing varies tremendously site-to-site. A recent report identified a novel motif, called the 3′GG motif, which substantially increases the efficiency of editing at all sites tested inC. elegans. Furthermore, they highlighted that previously published gRNAs with high editing efficiency also had this motif. I designed a Python command-line tool, ngg2, to identify 3′GG gRNA sites from indexed FASTA files. As a proof-of-concept, I screened for these motifs in six model genomes:Saccharomyces cerevisiae,Caenorhabditis elegans,Drosophila melanogaster,Danio rerio,Mus musculus, andHomo sapiens. I also scanned the genomes of pig (Sus scrofa) and African elephant (Loxodonta africana) to demonstrate the utility in non-model organisms. I identified more than 60 million single match 3′GG motifs in these genomes. Greater than 61% of all protein coding genes in the reference genomes had at least one unique 3′GG gRNA site overlapping an exon. In particular, more than 96% of mouse and 93% of human protein coding genes have at least one unique, overlapping 3′GG gRNA. These identified sites can be used as a starting point in gRNA selection, and the ngg2 tool provides an important ability to identify 3′GG editing sites in any species with an available genome sequence.
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ANURAGI, HIRDAYESH, AMBATI SRIJAN y BHARAT TAINDU JAIN. "RNA-guided multiplex genome engineering using cas9 nucleases for crop improvement: A review". Indian Journal of Agricultural Sciences 88, n.º 12 (11 de diciembre de 2018): 1811–17. http://dx.doi.org/10.56093/ijas.v88i12.85371.
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Crop improvement through genome editing techniques has aroused substantial excitement among agricultural scientists as it offers opportunities for precise and predictable modification directly in elite cultivars or accessions which are not possible by time-consuming conventional breeding schemes. CRISPR-Cas9, a recently evolved gRNA-guided nuclease technology, is crucial in plant genome modifications which enable us in carrying out rapid and extremely precise mutations at pre-determined genomic sites by using guide RNA which is complementary to the target locus.This targeted DNA is then cleaved by Cas9 nuclease to generate double-strand breaks (DSBs), followed by subsequent DNA repair via non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms leading to the disruption of gene functions in plants. Multiplexed CRISPR-Cas9 exploits multiple gRNA sequences for simultaneous editing of multiple sites within the same genome, demonstrating its efficiency over traditional gene pyramiding. CRISPR/Cas9 is relatively convenient and easier to implement technology that has proven to be extremely efficient and versatile as compared to previously developed tools like ZFNs and TALENs and hence, it has been considered as a revolutionary technique in crop genome editing. The technique is continuously evolving to produce variants with its application in enhancing the agricultural performance of most crops. Moreover, plants developed by this technique have been considered superior to transgenic plants due to non-involvement of foreign DNA, hence, alleviating safety issues associated with GM plants. The current review briefly emphasizes on the potentiality of this game-changing technology in revolutionizing the basic plant genetic research and plant breeding.
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Okada, Keita, Kanae Aoki, Teruyuki Tabei, Kota Sugio, Katsunori Imai, Yuki Bonkohara y Yusuke Kamachi. "Key sequence features of CRISPR RNA for dual-guide CRISPR-Cas9 ribonucleoprotein complexes assembled with wild-type or HiFi Cas9". Nucleic Acids Research 50, n.º 5 (15 de febrero de 2022): 2854–71. http://dx.doi.org/10.1093/nar/gkac100.
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Abstract Specific sequence features of the protospacer and protospacer-adjacent motif (PAM) are critical for efficient cleavage by CRISPR-Cas9, but current knowledge is largely derived from single-guide RNA (sgRNA) systems assessed in cultured cells. In this study, we sought to determine gRNA sequence features of a more native CRISPR-Cas9 ribonucleoprotein (RNP) complex with dual-guide RNAs (dgRNAs) composed of crRNA and tracrRNA, which has been used increasingly in recent CRISPR-Cas9 applications, particularly in zebrafish. Using both wild-type and HiFi SpCas9, we determined on-target cleavage efficiencies of 51 crRNAs in zebrafish embryos by assessing indel occurrence. Statistical analysis of these data identified novel position-specific mononucleotide features relevant to cleavage efficiencies throughout the protospacer sequence that may be unique to CRISPR-Cas9 RNPs pre-assembled with perfectly matched gRNAs. Overall features for wild-type Cas9 resembled those for HiFi Cas9, but specific differences were also observed. Mutational analysis of mononucleotide features confirmed their relevance to cleavage efficiencies. Moreover, the mononucleotide feature-based score, CRISPR-kp, correlated well with efficiencies of gRNAs reported in previous zebrafish RNP injection experiments, as well as independently tested crRNAs only in RNP format, but not with Cas9 mRNA co-injection. These findings will facilitate design of gRNA/crRNAs in genome editing applications, especially when using pre-assembled RNPs.
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Louie, Wilson, Max W. Shen, Zakir Tahiry, Sophia Zhang, Daniel Worstell, Christopher A. Cassa, Richard I. Sherwood y David K. Gifford. "Machine learning based CRISPR gRNA design for therapeutic exon skipping". PLOS Computational Biology 17, n.º 1 (8 de enero de 2021): e1008605. http://dx.doi.org/10.1371/journal.pcbi.1008605.
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Restoring gene function by the induced skipping of deleterious exons has been shown to be effective for treating genetic disorders. However, many of the clinically successful therapies for exon skipping are transient oligonucleotide-based treatments that require frequent dosing. CRISPR-Cas9 based genome editing that causes exon skipping is a promising therapeutic modality that may offer permanent alleviation of genetic disease. We show that machine learning can select Cas9 guide RNAs that disrupt splice acceptors and cause the skipping of targeted exons. We experimentally measured the exon skipping frequencies of a diverse genome-integrated library of 791 splice sequences targeted by 1,063 guide RNAs in mouse embryonic stem cells. We found that our method, SkipGuide, is able to identify effective guide RNAs with a precision of 0.68 (50% threshold predicted exon skipping frequency) and 0.93 (70% threshold predicted exon skipping frequency). We anticipate that SkipGuide will be useful for selecting guide RNA candidates for evaluation of CRISPR-Cas9-mediated exon skipping therapy.
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Louie, Wilson, Max W. Shen, Zakir Tahiry, Sophia Zhang, Daniel Worstell, Christopher A. Cassa, Richard I. Sherwood y David K. Gifford. "Machine learning based CRISPR gRNA design for therapeutic exon skipping". PLOS Computational Biology 17, n.º 1 (8 de enero de 2021): e1008605. http://dx.doi.org/10.1371/journal.pcbi.1008605.
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Restoring gene function by the induced skipping of deleterious exons has been shown to be effective for treating genetic disorders. However, many of the clinically successful therapies for exon skipping are transient oligonucleotide-based treatments that require frequent dosing. CRISPR-Cas9 based genome editing that causes exon skipping is a promising therapeutic modality that may offer permanent alleviation of genetic disease. We show that machine learning can select Cas9 guide RNAs that disrupt splice acceptors and cause the skipping of targeted exons. We experimentally measured the exon skipping frequencies of a diverse genome-integrated library of 791 splice sequences targeted by 1,063 guide RNAs in mouse embryonic stem cells. We found that our method, SkipGuide, is able to identify effective guide RNAs with a precision of 0.68 (50% threshold predicted exon skipping frequency) and 0.93 (70% threshold predicted exon skipping frequency). We anticipate that SkipGuide will be useful for selecting guide RNA candidates for evaluation of CRISPR-Cas9-mediated exon skipping therapy.
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Kato, Yuya, Hirotaka Tabata, Kumiko Sato, Mariko Nakamura, Izumu Saito y Tomoko Nakanishi. "Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient". International Journal of Molecular Sciences 22, n.º 19 (29 de septiembre de 2021): 10570. http://dx.doi.org/10.3390/ijms221910570.
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Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.
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Li, Qi, Bingbing Sun, Jun Chen, Yiwen Zhang, Yu Jiang y Sheng Yang. "A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli". Acta Biochimica et Biophysica Sinica 53, n.º 5 (25 de marzo de 2021): 620–27. http://dx.doi.org/10.1093/abbs/gmab036.
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Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (Cas9)-based genome editing tool pCas/pTargetF system that we established previously has been widely used in Escherichia coli MG1655. However, this system failed to manipulate the genome of E. coli BL21(DE3), owing to the potential higher leaky transcription of the gRNA-pMB1 specific to pTargetF in this strain. In this study, we modified the pCas/pTargetF system by replacing the promoter of gRNA-pMB1 with a tightly regulated promoter PrhaB, changing the replicon of pCas to a nontemperature-sensitive replicon, adding the sacB gene into pCas, and replacing the original N20-specific sequence of pTargetF with ccdB gene. We call this updated system as pEcCas/pEcgRNA. We found that gRNA-pMB1 indeed showed a slightly higher leaky expression in the pCas/pTargetF system compared with pEcCas/pEcgRNA. We also confirmed that genome editing can successfully be performed in BL21(DE3) by pEcCas/pEcgRNA with high efficiency. The application of pEcCas/pEcgRNA was then expanded to the E. coli B strain BL21 StarTM (DE3), K-12 strains MG1655, DH5α, CGMCC3705, Nissle1917, W strain ATCC9637, and also another species of Enterobacteriaceae, Tatumella citrea DSM13699, without any specific modifications. Finally, the plasmid curing process was optimized to shorten the time from $\sim$60 h to $\sim$32 h. The entire protocol (including plasmid construction, editing, electroporation and mutant verification, and plasmid elimination) took only $\sim$5.5 days per round in the pEcCas/pEcgRNA system, whereas it took $\sim$7.5 days in the pCas/pTargetF system. This study established a faster-acting genome editing tool that can be used in a wider range of E. coli strains and will also be useful for other Enterobacteriaceae species.
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Arakawa, Hiroshi. "A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism". Science Advances 2, n.º 8 (agosto de 2016): e1600699. http://dx.doi.org/10.1126/sciadv.1600699.
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The clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system is a powerful tool for genome editing that can be used to construct a guide RNA (gRNA) library for genetic screening. For gRNA design, one must know the sequence of the 20-mer flanking the protospacer adjacent motif (PAM), which seriously impedes experimentally making gRNA. I describe a method to construct a gRNA library via molecular biology techniques without relying on bioinformatics. Briefly, one synthesizes complementary DNA from the mRNA sequence using a semi-random primer containing a PAM complementary sequence and then cuts out the 20-mer adjacent to the PAM using type IIS and type III restriction enzymes to create a gRNA library. The described approach does not require prior knowledge about the target DNA sequences, making it applicable to any species.
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Nurkhasanah, Hidayati, Suharsono Suharsono y Tri Joko Santoso. "Construction and Introduction of CRISPR/Cas9-gRNA-Vinv for Editing A Gene Controlling Cold-Induced Sweetening [CIS] Character on Potato". Jurnal AgroBiogen 18, n.º 1 (15 de agosto de 2022): 21. http://dx.doi.org/10.21082/jbio.v18n1.2022.p21-32.
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<p>Cold-induced sweetening (CIS) causes the processed potato to have dark (browning) texture and induces high acrylamid formation. This phenomenon involves the activity of the Vacuolar invertase (Vinv) gene in accumulating sugar reducer in tubers under cold conditions. Inhibition of Vinv gene activity plays an important role in developing potato lines tolerance to CIS. The objectives of this study were to (1) construct a CRISPR/Cas9 cassette module carrying the gRNA from the Vinv gene, (2) obtain transgenic Atlantic potato cultivars containing CRISPR/Cas9-gRNA-Vinv cassette through genetic transformation by Agrobacterium tumefaciens, and (3) analyze the transgenic putative potato plant molecularly. The recombinant plasmid containing CRISPR/Cas9-gRNA-Vinv genes was successfully constructed and introduced into the potato genome by A. tumefaciens. Genetic transformation of Atlantic potato with CRISPR/Cas9-gRNA-Vinv cassette was more efficient using leaf explants compared to that using stem segment explants. Transformation of leaf explants resulted in transformation and regeneration efficiency values of 8.8% and 53.6%, respectively, compared to 1.3% and 0% transformation and regeneration efficiencies using stem segment explants. From 12 transformant lines obtained, three lines were found to contain the Cas9 gene. Sequence analysis of the Vinv gene derived from T0 transgenic plant, however, found no mutations occurred in the Vinv gene sequence. Further research is needed to obtain the Vinv gene mutants.</p>
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Zhang, Ping, Yu Wang, Chenxi Li, Xiaoyu Ma, Lan Ma y Xudong Zhu. "Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species". Journal of Fungi 7, n.º 7 (24 de junio de 2021): 505. http://dx.doi.org/10.3390/jof7070505.
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Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.
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Aguilar, Rhiannon R., Zih-Jie Shen y Jessica K. Tyler. "A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9". Methods and Protocols 5, n.º 5 (4 de octubre de 2022): 79. http://dx.doi.org/10.3390/mps5050079.
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Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome.
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Mubarok, Muhamad Husni, Atmitri Sisharmini, Aniversari Apriana, Tri Joko Santoso y Suharsono Suharsono. "(CRISPR/Cas9 Cassette Construction for OsARF2 Gene Editing and Development of Transgenic Rice Nipponbare Containing CRISPR/Cas9-OsARF2". Jurnal AgroBiogen 18, n.º 1 (4 de octubre de 2022): 45. http://dx.doi.org/10.21082/jbio.v18n1.2022.p45-56.
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<p>High Affinity K+ transporter 5 (HAK5) is one of the K + channel that involves in K + uptake. HAK5 gene expression is repressed under K + sufficient condition by the Auxin Response Factor 2 (ARF2) transcription factor. K + absorption can be increased by inactivation of ARF2 gene, e.g. by directed mutation using CRISPR/Cas9 method. The aims of this study were to create CRISPR/Cas9 construct carrying gRNA of OsARF2 gene and obtain transgenic rice Nipponbare containing CRISPR/Cas9- gRNAOsARF2. Two different sites in OsARF2 gene were succesfully designed as gRNA of the gene. Both oligo duplex RNA guides, gRNAOsARF2-A and gRNAOsARF2-B, were ligated into pDIRECT_21A plasmid using Golden Gate method. CRISPR/Cas9-gRNAOsARF2-A and CRISPR/Cas9-gRNAOsARF2-B cassettes were succesfully introduced separately into japonica rice cv. Nipponbare genome via Agrobacterium-mediated transformation, resulting putative transgenic lines. Analysis of Cas9 gene integration by PCR showed that 11 of 28 N-ARF-2-A putative transgenic lines contained Cas9 transgene. The sequencing analysis of the two selected lines showed that the OsARF2 gene of the CRISPR/Cas9-gRNAOsARF2 construct had not been mutated. Further studies are needed to determine the presence of mutations in the OsARF2 gene from other T0 transgenic lines obtained in this study together with that of the T1 transgenic lines.</p>
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Park, So Hyun, Ciaran M. Lee, Harshavardhan Deshmukh y Gang Bao. "Therapeutic Crispr/Cas9 Genome Editing for Treating Sickle Cell Disease". Blood 128, n.º 22 (2 de diciembre de 2016): 4703. http://dx.doi.org/10.1182/blood.v128.22.4703.4703.
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Abstract Introduction Sickle cell disease (SCD) is one of the most common monogenic disorders, affecting millions worldwide. SCD is caused by a point mutation in the β-globin gene (HBB). A single nucleotide substitution from A to T in the codon for the sixth amino acid in the β-globin protein converts a glutamic acid to a valine that leads to the production of sickle hemoglobin (HbS), which impairs the function of the red blood cells (RBCs). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only available cure, but it is feasible for only a small subpopulation (<15%) of patients and may be associated with a high risk. Here, we show that targeted genome editing can potentially provide a permanent cure for SCD by correcting the sickle mutation in clinically relevant hematopoietic stem and progenitor cells (HSPCs) for autologous transplantation. Methods For proof-of-concept, we designed CRISPR/Cas9 systems and donor templates to introduce the sickle mutation into wild-type (WT) HBB of mobilized peripheral blood CD34+ cells. To assess genome-editing outcomes mediated by CRISPR/Cas9 systems, we developed a novel digital droplet PCR (ddPCR) assay that can quantify the rates of non-homologous end joining (NHEJ) and homology directed repair (HDR) events simultaneously following the generation of DNA double strand breaks. The assay enables rapid and accurate quantification of gene modifications in HSPCs by CRISPR/Cas9 genome-editing. Specifically, Streptococcus pyogenes (Spy) Cas9 proteins, guide RNAs (gRNA), and single-stranded DNA (ssDNA) donor templates were delivered into CD34+ cells by nucleofection with optimized conditions. Different gRNAs targeting HBB near the SCD mutation site were tested, and the optimal gRNA was chosen based on high on-target activity and proximity to the mutation site. The optimal DNA donor design and concentration were determined based on the frequency of HDR events and viability/growth rate of edited cells. Treated samples and untreated controls were assayed as both single cell clones and in bulk culture. In 2-phase liquid culture, genome editing frequencies at both DNA and mRNA levels were quantified by ddPCR to confirm persistence of edited cells in the heterozygous population over time. The expression of globins and other erythroid markers were monitored using flow cytometry and real time PCR to determine if genome editing had any effect on the kinetics of erythropoiesis. Colony formation assays were used to determine the number and type of colonies following induction of differentiation. Colony ddPCR was performed to determine the genotype of edited cells. Wright/Giemsa stain was used to confirm terminal maturation of erythrocytes into enucleated RBC. Native polyacrylamide gel electrophoresis (PAGE) and high performance liquid chromatography (HPLC) were used to confirm translation of edited β-globin protein and formation of HbS. Results and Discussion We found that the efficiency of site-specific gene correction could be substantially improved by optimizing the CRISPR/Cas9 systems for genome editing. For example, with optimization, we achieved ~30% HDR rates in CD34+ cells with >80% cell viability. The HDR-modified alleles persisted in the population over the course of differentiation, and the edited CD34+ cells retained differentiation potential. Genotyping of individual erythroid colonies confirmed that up to 35% of colonies are either homozygous or heterozygous for HDR alleles. Following differentiation, treated cells express modified HBB mRNA and HbS. In addition, the off-target activity of the HBB-specific gRNAs was determined using both bioinformatics tools and unbiased genome-wide mapping techniques. Ongoing work includes the validation of gene correction in SCD patient derived HSPCs, characterization of modified cells in vitro and in vivo to assess the therapeutic potential, and analysis of long-term genotoxicity. Conclusions Based on the proof-of-concept study, we demonstrate that using the optimized CRISPR/Cas9 system and donor template, an HDR rate of ~30% can be achieved in CD34+ cells. The gene corrected cells have the potential to differentiate into erythroid cells that permanently produce WT β-globin. Our findings provide promising evidence for clinical translation of the HSPCs genome correction strategy in treating SCD patients, as well as correcting gene defects underlying other inherited single-gene disorders. Disclosures No relevant conflicts of interest to declare.
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Atmanli, Ayhan, Andreas C. Chai, Miao Cui, Zhaoning Wang, Takahiko Nishiyama, Rhonda Bassel-Duby y Eric N. Olson. "Cardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular Dystrophy". Circulation Research 129, n.º 6 (3 de septiembre de 2021): 602–16. http://dx.doi.org/10.1161/circresaha.121.319579.
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Rationale: Absence of dystrophin in Duchenne muscular dystrophy (DMD) results in the degeneration of skeletal and cardiac muscles. Owing to advances in respiratory management of patients with DMD, cardiomyopathy has become a significant aspect of the disease. While CRISPR/Cas9 genome editing technology holds great potential as a novel therapeutic avenue for DMD, little is known about the potential of DMD correction using CRISPR/Cas9 technology to mitigate cardiac abnormalities in DMD. Objective: To define the effects of CRISPR/Cas9 genome editing on structural, functional, and transcriptional abnormalities in DMD-associated cardiac disease. Methods and Results: We generated induced pluripotent stem cells from a patient with a deletion of exon 44 of the DMD gene (ΔEx44) and his healthy brother. We targeted exon 45 of the DMD gene by CRISPR/Cas9 genome editing to generate corrected DMD induced pluripotent stem cell lines, wherein the DMD open reading frame was restored via reframing or exon skipping. While DMD cardiomyocytes demonstrated morphological, structural, and functional deficits compared with control cardiomyocytes, cardiomyocytes from both corrected DMD lines were similar to control cardiomyocytes. Bulk RNA-sequencing of DMD cardiomyocytes showed transcriptional dysregulation consistent with dilated cardiomyopathy, which was mitigated in corrected DMD cardiomyocytes. We then corrected dysfunctional DMD cardiomyocytes by adenoviral delivery of Cas9/gRNA and showed that correction of DMD cardiomyocytes postdifferentiation reduces their arrhythmogenic potential. Single-nucleus RNA-sequencing of hearts of DMD mice showed transcriptional dysregulation in cardiomyocytes and fibroblasts, which in corrected mice was reduced to similar levels as wild-type mice. Conclusions: We show that CRISPR/Cas9-mediated correction of DMD ΔEx44 mitigates structural, functional, and transcriptional abnormalities consistent with dilated cardiomyopathy irrespective of how the protein reading frame is restored. We show that these effects extend to postnatal editing in induced pluripotent stem cell-derived cardiomyocytes and mice. These findings provide key insights into the utility of genome editing as a novel therapeutic for DMD-associated cardiomyopathy.
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Song, Letian, Jean-Paul Ouedraogo, Magdalena Kolbusz, Thi Truc Minh Nguyen y Adrian Tsang. "Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger". PLOS ONE 13, n.º 8 (24 de agosto de 2018): e0202868. http://dx.doi.org/10.1371/journal.pone.0202868.
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Dhokane, Dhananjay, Bhaskar Bhadra y Santanu Dasgupta. "CRISPR based targeted genome editing of Chlamydomonas reinhardtii using programmed Cas9-gRNA ribonucleoprotein". Molecular Biology Reports 47, n.º 11 (19 de octubre de 2020): 8747–55. http://dx.doi.org/10.1007/s11033-020-05922-5.
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Khan, Sikandar. "Recent Advancement and Innovations in CRISPR/Cas and CRISPR Related Technologies: A review". Biotechnology and Bioprocessing 2, n.º 5 (24 de junio de 2021): 01–12. http://dx.doi.org/10.31579/2766-2314/042.
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CRISPR genome editing technologies have been improving by every passing day. The initial CRISPR/Cas9 technologies, though emerged an improved version of genome editing in competition with TALENS and ZFNs, was nevertheless not free from technical and off-target effects. Technological improvements overtime start addressing issues with original CRISPR/Cas9 technology. The major areas of improvement targeted nucleases and delivery methods. Overtime the nuclease like Cas9 had some modifications like FokI-dCas9, Truncated guide RNAs (tru-gRNAs), Paired Cas9 nickase, Cpf1, Cas6 with Csm/Csr complex and chemically treated Cas9. In terms of delivery methods the improvements came along after almost all methods including viral methods like Recombinant Adeno Associated Viruses (rAAV), Lentivirus (LV), and bacteriophages. The review summarizes various non-viral gene delivery modes including physical methods like electroporation and chemical methods like nano particles, cell-derived membrane vesicles (CMVs) with upgraded developments. The review also compares various modes of delivering CRISPR gene editing machinery.
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Moniruzzaman, M., Yun Zhong, Zhifeng Huang y Guangyan Zhong. "Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis". International Journal of Molecular Sciences 23, n.º 9 (28 de abril de 2022): 4889. http://dx.doi.org/10.3390/ijms23094889.
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Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.
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Prasanth, Jagannadham y Thirugnanavel Anbalagan. "CRISPR-based genome editing: Catching impossibles for citrus improvements". International Journal of Agricultural and Applied Sciences 2, n.º 1 (30 de junio de 2021): 24–29. http://dx.doi.org/10.52804/ijaas2021.212.
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Citrus is globally one of the major fruit crops, occupying a place of prominence in international trade and tariff through horticultural commodities. Despite such distinction, citrus crop is confronted with a variety of biotic and abiotic stresses, thereby, sustaining production is always a daunting task. The genome size of citrus is rather small, ranging from 265 to 400 MB, probably an advantage for controlled trait specific editing. The evolution of next generation sequencing has facilitated the whole genome sequencing of as many 10 citrus species with 16 draft genome sequences, offering near future possibility to develop genome tailored citrus species or inducing the desired genetic transformation to address the issues chronically ailing commercial citrus cultivation in India, which is by no mean, a simple task to accomplish. Despite genetically intrinsic challenges involved in generating transgenics in perennial crop like citrus, several transgenics have been developed in namely, sweet orange, lemon, and grapefruit loaded with some useful traits. But, the public perception and the time taken to develop trangenics in citrus and less success ratio led the researchers adapt alternate ways. Of late, the thumping success of genome editing tools, especially Clustered. Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system has provided a new molecular tailoring machine for citrus improvement. In several citrus species like sweet orange, pummelo, and grapefruit CRISPR-Cas9 system has resulted in value added multiple traits-based transgenics. However, the major drawback of the CRISPR/Cas9 system is the generation of significant off-target cleavage sites as a result of complexing of gRNA with mismatched complementary target DNA within the genome. The use of CRISPR as genome editing technology is anticipated to induce many desired traits in citrus in years to come with more commercial applications in field for changed canopy structure, root traits, regular bearing, extended fruit maturity, besides multiple disease resistance.
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Kurniawati, Devi Ayu, NFN Suharsono y Tri Joko Santoso. "Editing of PCNA Gene by CRISPR/Cas9 Technology to Improve the Red Chili Resistance to Yellow Leaf Curl Disease". Jurnal AgroBiogen 16, n.º 2 (11 de diciembre de 2020): 79. http://dx.doi.org/10.21082/jbio.v16n2.2020.p79-88.
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<p>Yellow leaf curl disease caused by Pepper yellow leaf curl virus (PepYLCV), member of geminiviruses group, is responsible for heavy yield losses for chili pepper production. Resistant genes which can cause immunity to the disease have not been found in germplasm collection. The aim of the research was to edit proliferating cell nuclear antigen (PCNA) gene by using CRISPR/Cas9 technology for developing plant resistance against geminivirus in chili pepper. A CRISPR/Cas9 plasmid cassette construct harboring the guide RNA of PCNA gene was constructed by Golden Gate cloning strategy. The construct was then introduced into chili genome via in planta method using Agrobacterium tumefaciens EHA105. The transformed plants were bioassayed by virus inoculation and confirmed using PCR and DNA sequencing to identify a mutagenesis event in PCNA gene target. The results showed that CRISPR/Cas9 plasmid cassette harboring gRNA of PCNA gene was successfully constructed. In planta transformation using A. tumefaciens vector harboring CRISPR/Cas9-gRNA PCNA construct resulted in 307 and 193 transformed plants from chili var. Lingga and Ciko, respectively. Bioassay by using virus inoculation to the transformed plants obtained 6 and 14 lines of Lingga and Ciko, respectively, which were resistant to geminivirus (no symptom observed). The resistant lines of chili pepper var. Lingga and Ciko were mutated in PCNA gene with one base insertion or deletion mutation types. These results exhibit that the CRISPR/Cas9 genome editing can be used to induce mutant of PCNA gene in chili pepper. Further investigation is necessary to evaluate the selected chili lines resistant to PepYLCV infection.</p>
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Tang, Ning, Yumei Xia, Yijie Zhan, Junhao Dan, Mulan Yu, Xiaolan Bu y Mengliang Cao. "Improvement of Chloroplast Transformation Using CRISPR/Cas9". Journal of Biobased Materials and Bioenergy 14, n.º 3 (1 de junio de 2020): 401–7. http://dx.doi.org/10.1166/jbmb.2020.1970.
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Chloroplasts are organelles that contain genetic materials (DNA) in higher plant cells. The special genetic characteristics of chloroplasts mean that plasmid transformation has important research value, so it has become an important research direction second to nuclear transformation. Although the techniques of chloroplast genome modification have been successfully applied in tobacco and extended to other high plants, there are still many limitations. Exogenous genes are integrated into the chloroplast genome through homologous recombination. Therefore, the low efficiency of homologous recombination directly limits transformation efficiency. Gene editing with fixed-point cutting function and DNA damage repair mechanism may effectively improve the efficiency. In the present study, we aimed to use CRISPR/Cas9 to cut the site between two homologous recombinant fragments in chloroplast transformation to improve the efficiency by activating the DNA damage repair mechanism. The Cas9 gene and gRNA were added to the chloroplast transformation system of tobacco by co-transformation or integration into a transformation vector. The acquired resistant plants were screened by multiple selection of spectinomycin and chloroplast DNA was isolated for molecular detection by PCR. The results showed that the efficiency of chloroplast transformation increased by 6–10 times with the addition of gene editing technology. Although the transformation efficiency was still far below the level of nuclear transformation, this study may help to increase the efficiency of the plant chloroplast transformation system, and expand the types of plant receptors.
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Yang, Yue, Donghua Li, Fen Wan, Bohong Chen, Guanglan Wu, Feng Li, Yanliang Ren, Puping Liang, Jian Wan y Zhou Songyang. "Identification and Analysis of Small Molecule Inhibitors of CRISPR-Cas9 in Human Cells". Cells 11, n.º 22 (11 de noviembre de 2022): 3574. http://dx.doi.org/10.3390/cells11223574.
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Genome editing tools based on CRISPR–Cas systems can repair genetic mutations in situ; however, off-target effects and DNA damage lesions that result from genome editing remain major roadblocks to its full clinical implementation. Protein and chemical inhibitors of CRISPR–Cas systems may reduce off-target effects and DNA damage. Here we describe the identification of several lead chemical inhibitors that could specifically inhibit the activity of Streptococcus pyogenes Cas9 (SpCas9). In addition, we obtained derivatives of lead inhibitors that could penetrate the cell membrane and inhibit SpCas9 in cellulo. Two of these compounds, SP2 and SP24, were able to improve the specificity of SpCas9 in cellulo at low-micromolar concentration. Furthermore, microscale thermophoresis (MST) assays showed that SP24 might inhibit SpCas9 activity by interacting with both the SpCas9 protein and the SpCas9–gRNA ribonucleoprotein complex. Taken together, SP24 is a novel chemical inhibitor of SpCas9 which has the potential to enhance therapies that utilize SpCas9.
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Park, So Hyun Julie Park, Mingming Cao, Yankai Zhang, Vivien A. Sheehan y Gang Bao. "CRISPR/Cas9 Editing Induces High Rates of Unintended Large Gene Modifications in HSPCs from Patients with Sickle Cell Disease". Blood 138, Supplement 1 (5 de noviembre de 2021): 3969. http://dx.doi.org/10.1182/blood-2021-150739.
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Abstract Introduction: Several gene editing strategies have been developed to cure sickle cell disease (SCD), including the use of CRISPR/Cas9 to edit beta-globin (HBB), gamma-globin (HBG), or B-cell lymphoma/leukemia 11A (BCL11A) in hematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Although high gene-editing rates can be achieved and off-target effects reduced, new challenges in applying the gene-editing strategies, including unintended gene modifications, need to be addressed in order to cure SCD with high efficacy and safety. To date, due to limitations in sequencing methods, studies on CRISPR/Cas9 genome editing for treating SCD only identified small insertions/deletions (INDELs); the extent and consequences of unintended large gene modifications are generally unknown. Here we provide accurate quantification and profiling of unintended gene modifications due to Cas9 induced double-stranded breaks (DSBs) in SCD HSPCs, including large deletions, insertions, and complex chromosomal arrangements, and the comparison of different approaches. Methods: R-66S gRNA targets the sickle mutation on the HBB. R-02 gRNA generates a DSB 16 bp away from the sickle mutation site. SD-02 gRNA introduces a 13-bp Hereditary Persistence of Fetal Hemoglobin (HPFH) deletion as a major INDEL in the HBG1/HBG2 promoter to reactivate fetal hemoglobin (HbF). BCL11A gRNA targets the GATA1 site at the BCL11A erythroid enhancer to induce HbF. R-66S, R-02, SD-02, and BCL11A gRNAs were respectively complexed with SpyCas9 and delivered as ribonucleoprotein (RNP) to SCD HSPCs. To accurately quantify CRISPR/Cas9 induced large modifications in gene-edited SCD HSPCs, we used PacBio Single Molecule, Real-Time (SMRT) Sequencing with Unique Molecular Identifiers (UMI). The 5-6 kb region around the Cas9 cut-site was dual-UMI tagged using two PCR cycles. The second and third PCR was performed with minimal cycle numbers to enrich the UMI-tagged template molecules. The SMRTbell library composed of edited and unedited SCD HSPCs samples was sequenced on a PacBio Sequel II 8M flowcell using the circular consensus sequencing (CCS) mode. The PacBio subreads were converted to HiFi reads and subjected to UMI consensus read generation and variant calling. Results: SMRT-seq with UMI revealed high rates and broad spectra of unintended large deletions (> 200 bp) induced by Cas9 cutting at HBB, HBG1, and BCL11A genes in RNP treated samples, with respectively R-66S RNP, 31.7%; R-02 RNP, 17.4%; SD-02 RNP, 13.3%; BCL11A RNP, 40%. The large deletions have a very broad distribution of sizes and locations. In addition, we found large insertions (> 50 bp) and local complex chromosomal rearrangements at the Cas9 cut-sites. Therefore, the current assessment of gene-editing rates using short-read Next Generation Sequencing (NGS) misses a substantial proportion of Cas9-cutting induced large gene modifications, resulting in an inaccurate measure of both allele and genotype frequencies. Discussions: We found that unintended on-target large deletions occur at high rates at HBB, HBG1, and BCL11A in gene-edited SCD HSPCs. These results raise significant safety concerns regarding gene-editing of HSPCs to treat SCD. Our results demonstrate the importance of detecting and quantifying all possible CRISPR/Cas9 gene-editing outcomes to ensure the efficient and safe translation of gene-editing-based strategies to cure SCD and other human diseases. Additional work is required to determine the functional consequences of the unintended gene modifications and the persistence of the unintended large gene modifications at the on-target cut-sites. Disclosures Sheehan: Forma Therapeutics: Research Funding; Beam Therapeutics: Research Funding; Novartis: Research Funding.
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Riesenberg, Stephan, Nelly Helmbrecht, Philipp Kanis, Tomislav Maricic y Svante Pääbo. "Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage". Nature Communications 13, n.º 1 (25 de enero de 2022). http://dx.doi.org/10.1038/s41467-022-28137-7.
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AbstractThe first step in CRISPR-Cas9-mediated genome editing is the cleavage of target DNA sequences that are complementary to so-called spacer sequences in CRISPR guide RNAs (gRNAs). However, some DNA sequences are refractory to CRISPR-Cas9 cleavage, which is at least in part due to gRNA misfolding. To overcome this problem, we have engineered gRNAs with highly stable hairpins in their constant parts and further enhanced their stability by chemical modifications. The ‘Genome-editing Optimized Locked Design’ (GOLD)-gRNA increases genome editing efficiency up to around 1000-fold (from 0.08 to 80.5%) with a mean increase across different other targets of 7.4-fold. We anticipate that this improved gRNA will allow efficient editing regardless of spacer sequence composition and will be especially useful if a desired genomic site is difficult to edit.
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Allen, Daniel, Michael Rosenberg y Ayal Hendel. "Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells". Frontiers in Genome Editing 2 (28 de enero de 2021). http://dx.doi.org/10.3389/fgeed.2020.617910.
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CRISPR-Cas9 is quickly revolutionizing the way we approach gene therapy. CRISPR-Cas9 is a complexed, two-component system using a short guide RNA (gRNA) sequence to direct the Cas9 endonuclease to the target site. Modifying the gRNA independent of the Cas9 protein confers ease and flexibility to improve the CRISPR-Cas9 system as a genome-editing tool. gRNAs have been engineered to improve the CRISPR system's overall stability, specificity, safety, and versatility. gRNAs have been modified to increase their stability to guard against nuclease degradation, thereby enhancing their efficiency. Additionally, guide specificity has been improved by limiting off-target editing. Synthetic gRNA has been shown to ameliorate inflammatory signaling caused by the CRISPR system, thereby limiting immunogenicity and toxicity in edited mammalian cells. Furthermore, through conjugation with exogenous donor DNA, engineered gRNAs have been shown to improve homology-directed repair (HDR) efficiency by ensuring donor proximity to the edited site. Lastly, synthetic gRNAs attached to fluorescent labels have been developed to enable highly specific nuclear staining and imaging, enabling mechanistic studies of chromosomal dynamics and genomic mapping. Continued work on chemical modification and optimization of synthetic gRNAs will undoubtedly lead to clinical and therapeutic benefits and, ultimately, routinely performed CRISPR-based therapies.